Identification of genes required for mitosis in Drosophila

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IGP Genetics Mini-Symposium October 28th, 2005
Identification of genes required for mitosis in
Drosophila
Laura Lee Department of Cell and Developmental
Biology Vanderbilt University Medical Center
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Whats so great about flies?

• Cheap
• Short life cycle
• Rich tradition of genetics (100 years)
• Many genetic tools available
• Well-suited for large-scale genetic screens for
  mutations that affect a given process
• Post-genomics era
• Drosophila genome almost fully sequenced
• Innovative screening approaches
• Ideal simple model organism
• Many key biological processes/molecular pathways
  conserved in humans and flies
• Easier to unravel these processes in simple
  organisms

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Identification of genes required for mitosis in
Drosophila
awol
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Cell cycles of early Drosophila embryogenesis
key features
S
Male female pronuclei
13 cycles 2 hr
M
gt8,000 nuclei

• Rapid
• Driven by maternal stockpiles
• Syncytial nuclear divisions
• Posttranscriptional control

Claudio Sunkel
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Identification of Drosophila mitotic mutants
Focused on genes required for early embryogenesis

• Approach
• Maternal effect-lethal mutant collection
• Collected embryos from homozygous mutant females
• DNA staining microscopy
• Cell cycle defects observed?

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Identification of Drosophila mitotic mutants
Screened 2,300 maternal-effect lethal
lines (EMS-mutagenized) for mitotic defects
32 mutants/26 complementation groups

• Known cell cycle regulators identified
• 1 allele of grapes
• 2 alleles of giant nuclei
• 3 alleles of aurora A
• 1 allele of rough deal
• New cell cycle regulators identified
• 3 alleles of absent without leave
• 1 allele of no poles

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Largest complementation group on chrom. II (3
alleles)
Wild-type
Mutant
Arrest in metaphase-like state in cycles 1-7
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Genetic mapping of awol
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Candidate awol genes from genetic mapping
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All four alleles of awol contain mutations in
CG8981
Z2-1861
Q67
STOP
CG8981
?CG8981
1 kb
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Transgenic rescue of awol mutants
Hatch rate
Genotype
95.6
Wild-type
0
awol mutants
Conclusion CG8981 is the awol gene
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Identification of genes required for mitosis in
Drosophila
awol
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IVEC Screen (In Vitro Expression Cloning)
cDNA Library
Bacterial Colonies
Plasmid Pools
35S-labeled Protein Pools
Biochemical Screens (e.g. identification of
kinase or protease substrates)
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Drosophila Gene Collection

• Established by Berkeley Drosophila Genome Project
• Goal Each Drosophila gene represented by an
  individual full-length cDNA
• Release 1 consists of 6,000 full-length cDNA
  clones representing 42 of Drosophila genome
• cDNA clones are arrayed (17 x 384 well plates)

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Examples of 35S-labeled protein pools from
Drosophila Gene Collection
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Ooi/Kirschner lab
DIVEC screen to identify substrates of the
Anaphase-Promoting Complex (APC)
APC promotes ubiquitin-mediated proteolysis of
key cell cycle regulators -gt Exit from mitosis
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DIVEC screen for APC-Cdh1 substrates
Cdh1-dependent Degradation
(Ooi/Kirschner)
Protein (novel) p71 p78 p91
AWOL (Human AWOL)
Localization
Control
Cdh1
Microtubules
Cytoplasmic
Nuclear
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Drosophila and human AWOL proteins contain BRCT
domains
female
Drosophila
male
human
BRCT domains are protein- and DNA-binding domains
found in many proteins involved in cellular
response to DNA damage
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AWOLDrosophila homolog of the human
microcephalin gene
Disease gene autosomal recessive primary
microcephaly
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Use of Drosophila in the study of human disease
BLAST search of 929 human disease genes found
that 77 (714 genes) have readily identifiable
homologs in Drosophila Ease of performing
genetic screens in Drosophila allows one to
readily place human disease genes within
molecular pathways
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Microcephalin protein has undergone rapid
evolution
Contributed to brain expansion along primate
lineage leading to humans
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(No Transcript)
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What is the normal cellular/molecular function of
Microcephalin?

• Role in cell cycle? Hypothesized to regulate
  mitotic divisions in vertebrate brains -gt
  adequate cell
• Role in DNA damage response? (contains BRCT
  domains)

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Wild-type
awol
Are abnormal mitotic spindles centrosomes in
awol mutants due to activation of a Chk2 pathway?
DNA Alpha-Tubulin
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Chk2-mediated centrosomal inactivation in early
Drosophila embryos
DNA damage/incomplete replication Chk2
activation Mitotic spindle/centrosome
defects Mitotic arrest Removal of defective
nuclei Purpose Safety mechanism to prevent
propagation of damaged/incompletely replicated DNA
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Are abnormal mitotic spindles centrosomes in
awol mutants due to activation of a Chk2 pathway?
Rationale if abnormal mitotic spindles and
centrosomes in awol mutants are due activation of
the Chk2 pathway, then LOSS of Chk2 should
PREVENT formation of abnormal spindles and
centrosomes in awol mutants Genetic experiment
create double mutants, look at phenotype
Conclusion the Chk2 pathway is activated in awol
mutants

• Why? Incomplete DNA replication and/or DNA damage?

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Hydrodynamic studies of AWOL protein by sucrose
density centrifugation
High MW (300-400 kDa)
Monomer
Dimer?
(Top)
(Bottom)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
16 17 18 19 20 21
Myc Western blot
Load Extracts of embryos expressing Myc-AWOL
transgene
Conclusion AWOL exists in a large MW complex in
Drosophila embryos

• What else is in the complex?

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Conclusions

• We have identified mutations in awol, the
  Drosophila homolog of the human disease gene,
  microcephalin
• AWOL is required for mitosis in the early embryo
• AWOL is a substrate of the Anaphase-Promoting
  Complex, a major cell cycle regulator
• The checkpoint pathway mediated by Chk2 is
  activated in awol mutants
• Future efforts will focus on placing AWOL in a
  molecular pathway using genetics and biochemistry
• Understanding how AWOL functions will give us
  insight into human brain development and evolution

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Acknowledgments
Lee Lab members My lab Ethans
lab Joint Michael Anderson Chris
Cselenyi Jun Dou Jessica Keel Kristin
Kalie Audrey Frist Julie Merkle
Emilios Tahinci Erin Loggins
Curtis Thorne
Female-sterile mutants Charles Zukers lab
Jamie Rickmyre
Zuker mutant screen Terry Orr-Weaver
Lab (Whitehead Institute)
APC substrates Danny Ooi/Marc Kirschner (Harvard
Medical School)